ProjectSummary Cells are highly dynamic, squeezing, pulling, and tugging on their surroundings and on each other. Each individualinteractioninvolvesforces.Theseforcesarefeltbyspecificreceptorsandmolecules.Althoughsmall inmagnitude(pN),thesemolecularforcescanhaveprofoundbiologicalimpactsinmanyaspectsofcellularlife includingthefateofdifferentiatingstemcells,celldivision,cancermetastasis,andbloodclotting.Therefore,the ability to characterize the interplay between physical forces and biochemical signals isa critical component of understanding signaling pathways in living systems. There are two main techniques used to study molecular mechanobiology: single molecule force spectroscopy (SMFS) and traction force microscopy (TFM) based methods.Whilepowerful,theseapproachessufferfromseveraldrawbacks.SMFSmeasuresindividualreceptor forces(pN),butitdoessoonlyonemoleculeatatime.ConverselyTFMprovidesspatialmapsofcellularforces, butonthenNscale,ordersofmagnitudelargerthantheforcesappliedbyindividualcellreceptors.Tobridge these approaches, we invented molecular tension fluorescence microscopy (MTFM) which uses conventional fluorescencemicroscopytomapcellularforceswithpNresolutionbyusingacalibratedmolecularforceprobe. ThegoalofthisproposalistotransformthecapabilitiesofMTFMallowingordersofmagnitudeimprovementin spatialandtemporalresolutionaswellasthemappingofforceorientation.Molecularmechanobiologyremains at the fringes of biomedical sciences because of the lack of tools to precisely quantify and link mechanics to cellularbiochemistry.Ourgoalistotransformthefieldofmolecularmechanobiologybydevelopingnewimaging technologies to enable the study cellular forces at unprecedented resolution. These technologies, centered around the DNA-based MTFM probes, will provide a broadly applicable platform of technology to investigate molecularmechanics,andthefunctionaloutcomesofmolecularforces,indiversebiologicalsystems. InAim1 wewilladdressthespatialresolutiongap,andleveragetheDNA-basedforceprobestodevelopsuper-resolution force-PAINT with the goal of dynamic force imaging with 20 nm spatial resolution. In Aim 2 we will probe the dynamicsofforcesandforcefluctuationsbyharnessingthepoweroftwoapproaches,FRAPandFCS,tostudy molecular force dynamics with nsec to msec time resolution. Finally, in Aim 3 we will leverage fluorescence polarization microscopy to measure the 3D orientations of molecular forces. We will use fibroblast focal adhesions, platelet activation and coagulation, and T cell antigen recognition to test and verify our approach. Accomplishmentofthesegoalswillprovideanewtoolkitforunderstandingmolecularforcesandgeneratinga frameworkofhowforceorganizationanddynamicsinfluencecellularfunctioninhealthyanddiseasestates.